1-Oxylimidazolinyl compounds used as stable free radical pH indicators

ABSTRACT

1-Oxylimidazolinyl compounds bridged to an asymmetric center by a methylene group at the two position of the ring find use in determining acidity or basicity of a medium. One of the groups bonded to the asymmetric carbon atom is capable of gaining or losing a proton which results in a change of asymmetry about the carbon atom. Because the environment of the two protons of the methylene group is different by virtue of the asymmetry of the adjacent carbon atom, changes in asymmetry about the asymmetric carbon atom, change the electron spin resonance spectrum of the compound. The changes in the electron spin resonance spectrum can be related to the acidity or basicity of the medium.

United States Patent 1 Becher et al.

[ 1 Dec. 16, 1975 l l I-OXYLIMIDAZOLINYL COMPOUNDS USED AS STABLE FREERADICAL PH INDICATORS [75] Inventors: Jan Becher, Grindlosc; Edwin F.Ullman, Atherton, both of Calif.

Related US. Application Data [60] Division of Ser. No; 117,669, Feb. 22.1971, Pat. No. 3,706,537, which is a continuationin-part of Ser. No.794,008, Jan. 27, :969, abandoned.

[52} US. Cl...... 260/309.6; 23/230 R; 260/256.4 C; 260/257; 260/276 R;260/297 R [51] Int. Cl. C07D 233/22 [58] Field of Search 260/309.6

[56] References Cited UNITED STATES PATENTS 3,697,538 10/1972 Boocock etal 260/309.6 3,732,244 5/1973 Boocock et al 2150/3096 OTHER PUBLICATIONSBox et al. Chem. Abst. Vol. 66, No. l000l9u (1967).

Boocock et al. J. Amer. Chem. Soc. ol. 90. pp. 5945-5946 (1968).

Boocock et al. J. Amer. Chem. Sc; 68736874 (1968).

Osiecki et al. J. Amer. Chem. r. .c. Vol. 90, pp. 1078-1079 (1968).

oi 90, pp.

Primary ExaminerNatalie Trousof Attorney, Agent, or FirmTownsend andTownsend ABSTRACT l-Oxylimidazolinyl compounds bridged to an asymmetriccenter by a methylene group at the two position of the ring find use indetermining acidity or basicity of a medium. One of the groups bonded tothe asymmetric carbon atom is capable of gaining or losing a protonwhich results in a change of asymmetry about the carbon atom. Becausethe environment of the two protons of the methylene group is differentby virtue of the asymmetry of the adjacent carbon atom, changes inasymmetry about the asymmetric carbon atom, change the electron spinresonance spectrum of the compound. The changes in the electron spinresonance spectrum can be related to the acidity or basicity of themedium.

2 Claims, No Drawings l-OXYLIMIDAZOLINYL COMPOUNDS USED AS STABLE FREERADICAL PH INDICATORS BACKGROUND OF THE INVENTION 1. Field of theInvention The determination of acidity is important in numerous systems.In processing, the acidity of the medium may be essential to the yieldof the product, the course of the reaction or the determination of thedegree to which the reaction has occurred. In many instances, materialsare present which will interfere with an electro-chemical determinationof the acidity of the system.

ESR spectrometry can be employed with opaque solutions, where opticalmethods cannot be employed. In addition, free radical compounds can beemployed in situations where it might be difficult or otherwiseimpossible to introduce a probe to determine the acidity of the system.For example, appropriately substituted compounds could be introducedinto individual cells and measurements made on the cells, withoutdestruction of the cell.

2. Description of the Prior Art Co-pending application Ser. No. 696,718,filed Jan. 10, 1968, now abandoned, discloses tetra-substitutedimidazolidinyl-l-oxyl-3-oxide compounds which are stable free radicals,having a wide variety of substituents at the 2 position of the ring.Co-pending application Ser. No. 752,744, filed Aug. 15, 1968, nowabandoned, discloses tetra-substituted imidazolidinyl-l-oxyl compoundswhich are also stable free radicals. These compounds have been taught tobe useful as spin labels and as standards for ESR spectrums.

DETAILED DESCRIPTION Compounds are provided which permit aciditydeterminations by measuring variations in electron spin resonancespectra. The salient features of the subject invention are the presenceof (1) a center which changes its degree of asymmetry by the loss orgain of a proton which is (2) bonded to a methylenic group, which is (2)bonded to a functionality having an unpaired electron.

The compounds of this invention are primarily anitronyl nitroxides ora-imino nitroxides having a methylene (CH group bonded to the carbonatom intermediate the two nitrogen atoms. To the other valence of themethylene is a group which with change in acidity (basicity) undergoes achange in symmetry. That is, the methylene and the three groups providea center, which loses, gains or changes the asymmetry about the centralcarbon atom. Since the unpaired electron resonates between the variousatoms of the nitronyl nitroxide and imino nitroxide, the electron spinresonance spectrum of the molecule is influenced by the fields of thetwo protons of the methylene group. By affecting the equivalence of thetwo protons by the presence of an asymmetric center, changes in theelectron spin resonance spectrum can be observed.

The subject compounds have the nitrogen atoms bonded to carbon atomswhich are bonded to three other carbon atoms, preferably so as to form afive 2 membered ring which is disubstituted in both the 4 and the 5positions.

Preferably, the compositions of this invention will have a rigid ringstructure of the following formula:

wherein R R are organic radicals, preferably hydrocarbon radicals, offrom 1 to 20 carbon atoms, more usually from 1 to 12 carbon atoms, whichmay be aliphatic, alicyclic, both either saturated or aliphaticallyunsaturated, i.e., ethylenic or acetylenic, usually not more than onesite of unsaturation, aromatic, or combination thereof, and are bondedto the carbon atoms of the ring through carbon.

The RR may be the same or different, but because of preparativeconvenience, Il -R will usually be the same as R R Furthermore, two ofthe RR" may be taken together to form a ring, usually of from 5 to 7carbon atoms. If RR is taken together, a ring spiro to the imidazolidinering will be obtained. If R and R are taken together, a ring fused tothe imidazolidine ring will be obtained. The sole significant factorconcerning R R are that they are organic radicals bonded to the ringcarbon atoms through carbon.

W is oxide (O), n is an integer of from 0 to 1, with the proviso thatwhen n is l the nitrogen atom to which W is bonded is positive, and X, Yand Z represent at least two separate groups, usually hydrogen, organicor an organic functionality, at least one of which has or at least oneis an acidic or basic substituent, which gains or loses a proton withchange in acidity of the medium into which it is introduced; X, Y and Zbeing selected so that the degree of asymmetry about C* changes with thegain or loss of a proton by said acidic or basic group.

When n is 0, the compounds will have the following formula:

wherein all of the symbols are defined previously.

When n is l, the compounds will have the following formula:

wherein all of the symbols have been defined previously.

Preferred compositions are those having R R alkyl, aryl hydrocarbon oraralkyl of from 1 to 12 carbon atoms, more preferably alkyl from 1 to 6carbon atoms, and particularly preferred methyl or ethyl.

The total number of carbon atoms in the molecule, will usually notexceed 100 carbon atoms and more usually not exceed 60 carbon atoms,preferably being of from about to 50 carbon atoms.

As already indicated, the significant features concerning X, Y and Z arethat the degree of asymmetry about C* varies with change in acidity. Itis found that the farther one removes the site which gains or loses theproton from the methylenic group the greater the spectral changes in theESR are attenuated. However, if the geometry is such, that the acidic orbasic group is within approximately 6 A, there will be sufficient changein the spectrum to permit an accurate measurement. Accordingly, allsubstituents are contemplated on the beta carbon atom in which at leastone of the atoms which gains or loses a proton with change in acidity iscapable of approaching to within 6 A of the methylenic group (alphacarbon atom) or the unpaired electron containing functionality, i.e.,nitronyl nitroxide or imino nitroxide.

Usually the substituents on the beta carbon atom and particularly thosecontaining the acidic or basic entity which donates or loses a proton,should be attached to relatively short molecular chains. For example,this is borne out in the fonnula given above, if Y is hydrogen and X ismethyl; the observable ESR spectral changes are attenuated in the seriesin which Z is COOH, CH COOH, CH CH COOH, etc.

However, the length of the chain to which the acidic or basic centersare attached is not controlling in all cases. Special sten'c effects inrigid groups can serve to bring the active center spatially near thenitronyl nitroxide group despite the fact that the acidic or basic groupis many atoms removed. For example, a substituent on the beta carbonatom such as:

HOOC

would provide the desired change in asymmetry that is observable by ESRspectroscopy sufficiently for present purposes. The groups bonded to thebeta carbon atom are therefore not strictly limited by molecular sizebut only by spatial atomic orientation.

In addition, the groups X, Y and Z may be bonded together to form ringswhere the asymmetric center is the bridge head carbon in a bicyclic ringor otherwise, an annular carbon atom. In this situation also, the degreeof asymmetry must change with a gain or loss of a proton.

With the above spatial and symmetry requirement the only otherrequirement for the substituents on the beta carbon atom is the presenceof at least one acidic or basic group. Any group capable of gaining orlosing a proton in acidic or basic media is contemplated. Typical acidicgroups include phenols, carboxylic acids, barbituric acids, SO H,OSO;,H, PO H O- PO H -SO H, OPO H,

4 CH(NH COOH and the like. Typical basic groups include amines,heterocyclic bases such as pyridine and quinoline, amidines -C(=Nl-l)NHquinolines, -NHC(=Nl-I)NH and the like.

The nature of the groups on the beta carbon atom, aside from thenecessary basic or acidic entity, may be as diverse as desired.Preferably these groups should be as different from one another aspossible both with respect to size and to polarity. The groups may bealiphatic or aromatic and may contain any functional groups. Only thechange in asymmetry about the beta carbon atom with the gain or loss ofproton is critical with regard to this portion of the total free radicalmolecule.

Preferred compounds will have X equal to hydrogen or lower alkyl; Y willbe hydrocarbon or heterohydrocarbon having from 0 to 3 heteroatoms e.g.sulfur, oxygen, nitrogen, halogen, etc., will be different from X andusually of from 1 to 20 carbon atoms; Z will have the basic or acidicfunctionality and will be from 0 to 20 carbon atoms.

Organic free radical compounds which come within the subject inventioncan have groups bonded to the methylene group of the following formulae:

wherein X is hydrogen or lower alkyl of from 1 to 6 carbon atoms;

carbon atoms, Y is different from X' and is lower alkyl, alkoxycarbonylor carboxylic acid; and

wherein X is hydrogen or lower alkyl and Y is different from X and islower alkyl.

Alternatively, Y and Z may be taken together to form a ring of from 5 to7 annular members, which have 1 to 3 heteroannular members e.g. oxygenor nitrogen, whereby the degree of asymmetry changes with the loss orgain of a proton.

In referring to the measuring of acidity, it is also intended to includemeasurements of basicity. That is, depending upon the compounds employedin this invention, the degree to which a compound accepts or donates aproton in a particular medium from or to the free radical compoundemployed in the subject invention can be related to extrinsic standards.

.benzene, chloroform, chlorobenzene and miscellaneous solvents such asnitrobenzene, dimethylformamide, hexamethyl phosphoramide, acetonitrile,dimethyl sulfoxides, etc. In non-aqueous solvents, the acidity orbasicity is not properly referred to as pH, but rather is more relatedto the ability of the solvent to accept or donate a proton.

The change in spectrum is to a significant degree based on the change insolvation of the group which gains or loses a proton. Preferred media,therefore, will be those which provide strong solvation of the charge.These are normally hydroxylic media, such as water and the alcohols.

The change in spectrum will be most pronounced where there is thegreatest change in concentration differential between the protonated andunprotonated species. That is, the pK of the radical compound should besuch that a substantial amount of the compound should be in both theprotonated and uprotonated form. Usually, both of the forms should bepresent in at least 5 molar percent and preferably to at least molarpercent.

The spectral changes observed with change in symmetry about the betacarbon atom will vary with the particular radical being used and theenvironment being analyzed. With the nitronyl nitroxide, generally, thechanges in the spectrum are from five groups of three lines each to fivegroups of four lines each. With the imino nitroxide, the change in thespectra will vary depending upon whether the imino nitrogen becomesprotonated. The imino nitroxide will have a more complex spectrum thanthe nitronyl nitroxide depending on the groups associated with theunpaired electron and the medium being measured.

In some cases a change may not be complete. For example, the three linegroups may change only by broadening of the center line or the four linegroups may change only by partial shifting of the two center linestoward each other. Those radicals which give a pronounced and completechange will usually be more desirable, where the most accurate andprecise determinations are desired.

The accuracy of the determination will be solely dependent on the degreeof change in the ESR spectrum with the gain or loss of a proton by thefree radical compound. Since, at the equivalence point half of the freeradical compound will be in the protonated form and half will be in theunprotonated form, the spectrum, which normally will be taken near theequivalence point, will be a composite of the neutral and ionic forms.Since extrinsic standards will be employed for the determination of pH,it is only necessary that the change in the ESR spectrum vary in asmooth way with the change in the acidity or basicity of the medium.

The compositions of this invention are readily prepared employing analdehyde having the appropriate asymmetric center in the beta positionand a l,1,2,2- tetrasubstituted l,2-bis-hydroxylaminoethane. Thesubstituents at the l and the 2 position are those which have beendescribed previously as RR The resulting 1,3-dihydroxydiazole may beused to form the nitronyl nitroxide or the imino nitroxide byappropriate methods. See co-pending applications, Ser. Nos. 740,055filed June 26, 1968, now abandoned, and Ser. No. 752,744 filed Aug. 15,1968, both now abandoned.

The following examples illustrate the preparation of typical compoundsof the present invention. In the structural formulae, the symbol Thefirst illustration utilizes a scheme of synthesis as follows:

SCHEME A CQOC H arcu cn (oc n 2 c Base R COOC H is used to indicate 1)NHOH NHOH R )1 2 OXIDATION Three compounds (lVa, IVb, lVc) of thisinvention are obtained by the above synthesis in which the substituent Rof the product IV has the following meaning (corresponding intermediatesare similarly identified by the letters a, b, and 0.):

IV a: R CH, IV b: R C H 5-Methyl-5-(formylmethyl)-barbituric acid III a(All temperatures here and throughout this specification are inCentigrade.)

The malonic ester I a (10.0 g., 0.0345 mol) and urea (2.63 g., 0.0438mol) were added to a solution of sodium 1.83 g., 0.0794 mol) in 48.5 ml.dry ethanol. The

solution was gradually concentrated to 12 ml. by slow distillation over2 hours and then heated at 85 for 4 hours, followed by stirring at roomtemperature for 12 hours. The semicrystalline reaction mixture was thencooled to and 39 ml. ice cold water was added. The water solution wasextracted with benzene (2 X 14 ml.), the combined benzene extractswashed with a little water and the combined water phase was acidifiedwith 8N HCl. After stirring some time at 5, 5.7 g. (64%) of5-methyl-5-(formylmethyl)-barbituric acid diethyl acetal II a hadprecipitated. A sample recrystallized from benzene melted at l00110.

The acetal II a (1.90 g., 0.00736 mol) was refluxed for 1 hour in 23 ml.of 0.067 N HCl and then cooled with stirring to 0. The precipitatedwhite crystals were washed with a little ice cold water andrecrystallized from ethanol to give 0.87 g. (65%) of5-methyl-5-(formylmethyl)-barbituric acid 111 a, m.p. 247-249 d.

Anal. Calculated for C H N O C, 45.65; H, 4.38; N, 15.21. Found: C,45.56; H, 4.49; N, 15.38.

5-Methyl-5-(1,3-dioxy-4,4,5,5tetramethyl-4,5'dihydro-2-imidazomethyl)-barbituricacid IV a The aldehyde III a (0.5 g., 0.0027 mol) was mixed with2,3-bis-hydroxylamino-2,3-dimethylbutane (0.41 g., 0.0028 mol) in ml.absolute ethanol. The solution was stirred at room temperature for 3 /2hours. Then 5.0 g. lead dioxide was added and stirring continued for 10min. The deep red reaction mixture was filtered through Celite,evaporated in vacuo and the residue chromatographed on silica with a 9:1 mixture of chloro-form-methanol. The main red band yielded 0.22 g,(26%) of the barbituric acid radical IV a. A sample (0.22 g.)recrystallized from methanol-ether gave deep red needles (0.15 g.), m.p.213-216 d.

Anal. Calculated for C H N O C: 50.1, H: 6.1, N: 18.0 Found: C: 50.26,H: 5.97, N: 17.96.

5-Ethyl-5-(formylmethyl)-barbituric acid 111 b The malonic ester I b(8.0 g., 0.029 mol) and urea (2.21 g., 0.037 mol) were added to asolution of sodium (1.53 g., 0.067 mol) in 41 ml. of dry ethanol undernitrogen. 33 ml. of ethanol was then distilled off over 5 hours at whichtime the temperature of the reaction mixture was 86. The remainingreaction mixture was cooled to 10 and diluted with 24 ml. of ice coldwater. The water solution was extracted with benzene (2 X 12 ml.) andthe combined benzene extracts washed with a little water. The combinedwater phase was brought to pH 3.1 by adding 8 N HCl. White crystalsprecipitated after stirring some time with ice cooling.

Yield: 5.8 g. (74%) of the acetal II b, m.p. 146-149 d The acetal II b(5.0 g., 0.021 mol) was refluxed for 1 hour in 60 ml. 0.067N HCl andthen cooled to 0 with stirring. The precipitated white crystals werefiltered, washed with a little ice cold water and dried to give 3.0 g.(74%) of the aldehyde 111 b, m.p. 256-259 d. A sample recrystallizedfrom 0.1N HCl had m.p. 25625 8 d., colorless needles.

Anal. Calculated for C H N O C: 48.48; H: 5.09; N: 14.14. Found: C:48.28; H: 5.17; N: 14.30.

5-Ethyl-5-( l ,3-dioxy-4,4,5 ,5-tetramethyl-4,5-dihydro-Z-imidazomethyl)-barbituric acid IV b The aldehyde 111 b (0.1 19 g.,0.6.10 mol) in 5.0 ml. absolute ethanol was mixed with2,3-bis-hydroxylamino-2,3-dimethylbutane (0.089 g., 0.6 X 10' mol) in5.0 ml. benzene and stirred for 4 /2 hours at room temperature afterwhich time all the starting material had dissolved. Then 2.0 g. leaddioxide was added and stirring continued for another /2 hour. The deepred reaction mixture was filtered over Celite, evaporated in vacuo andthe residue chromatographed on silica with a 10:1 mixture ofchloroform-methanol. The main red band yielded on evaporation in vacuo0.128 g. (66%) of the barbituric acid radical IV b. A sample wasrecrystallized from chloroformether, m.p. 191l94 c.

Anal. Calculated for C, H N O C: 51.7; H: 6.5; N: 17.2 Found: C: 51.42;H: 6.33; N: 17.13.

Diethyl (2,2-diethoxyethyl)-( l-methylbutyl)-malonate I c Sodium hydride(50% suspension in oil, 8.8 g., 0.183 mol) was washed with dry benzeneand suspended in ml. dry dimethylformamide under dry nitrogen. To thissuspension was added dropwise diethyl (l-methylbutyl)-malonate (44.0 g.,0.191 mol), while the temperature was maintained at 4050.

When the addition was complete and no more hydrogen was evolved,bromoacetaldehyde diethylacetal (45.2 g., 0.229 mol) was added and themixture was heated at 123 for 30 hours with stirring under dry nitrogen.The mixture was then concentrated in vacuo, cooled to 15, and dilutedwith50 ml. of water. The water solution was extracted with ether (3 X100 ml.), the combined extracts dried over magnesium sulfate .andconcentrated in vacuo. The residual light yellow oil was distilled togive 29.9 g. (46%) of l c, b.p. 102105/0.05 mm. Hg.

5-(1-Methylbutyl)-5-(formylmethy1)-barbituric acid 111 The malonic ester1 c (11.85 g., 0.0345 mol) and 2.62 g. of urea (0.044 mol) was added toa solution of sodium (1.83 g., 0.0795 mol) in 49 ml. dry ethanol underdry nitrogen and 37 ml. was gradually distilled off over 2 hours. Thetemperature of the reaction mixture rose to 95 during this distillation.The reaction mixture was then stirred at 90 for 6 hours and at roomtemperature for another 15 hours. After cooling to 10, ice water wasadded and the solution was extracted with ether (2 X 10 ml.). Thecombined ether extracts were washed with a little water and the combinedwater phase was brought to pH 2.1 by adding 6N HCl, whereupon an oilseparated. The water phase was then extracted with chloroform (4 X 100ml.) and the extracts dried over magnesium sulfate and evaporated invacuo to give a colorless oil which crystallized upon scratching. Dryingover P gave 8.78 g. (81%) of the acetal II c, m.p. 6369.

The acetal (c 95.0 g., 0.0159 mol) was refluxed for 1 hour in 48 ml.0.067N l-lCl. After cooling slowly to 0 the solution deposited whitecrystals.

Yield: 2.6 g. (68%) of the aldehyde 111 c, m.p.-

l28-132. A sample recrystallized twice from 0.1 N HCl had m.p. 161-162(softening at 145).

Anal. Calculated for C H N O C: 54.99; H: 6.71; N: 11.66. Found: C:54.78; H: 6.93; N: 11.48.

5-( 1-Methylbutyl)-5-( 1,3-dioxy-4,4,5,S-tetramethyl-4,5-dihydro-2-imidazomethyl)-barbituric acid IV c The aldehyde 111 c(3.0 g., .0125 mol) was mixed with2,3-bis-hydroxylamino-2,3-dimethylbutane (2.32 g., 0.0156 mol) in 325ml. benzene and stirred for 3 days at room temperature. The benzenesolution was then evaporated in vacuo and the residue dissolved (in 100ml. ethyl acetate). After drying and with magnesium sulfate andevaporation in vacuo (4.40 g. (95%) of pink crystal were obtained. 1.0g. (0.00271 mol) of this crude adduct was stirred in 250 ml. .1 N sodiumhydroxide with sodium periodate (0.87 g., 0.00271 mol) at 0 for 8 min.The deep red solution was then brought to pH 7.0 by adding 1 Nhydrochloric acid and extracted with chloroform (4 X 100 ml.). Afteraddition of another 10 ml. hydrochloric acid, the water phase was againextracted with 100 ml. chloroform. The combined chloroform solutionswere dried over magnesium sulfate, filtered and evaporated in vacuo. Theresidue was chromatographed on silica with dry ether. Theproductappeared as a strong red band which was collected. After concentrationof this fraction and cooling, red crystals precipitated.

Yield: 0.418 g. (40%) of IV c, m.p. l88l9l. Anal. Calculated for C I-1 N0 C: 55.60; H: 7.35; N: 15.26. Found C: 55.80 H: 7.24; N: 15.17.

The next sequence of experimental work utilizes the reaction scheme asfollows to make the product VIII of this invention:

methyl-( 1 ,3-dioxyl ,1 ,5,5-tetramethyl-4,5-dihydro-2-imidazomethyl)-malonate VII The malonic ester 1 a (4.0 g., 0.0138 mol)was refluxed in 20 ml. 0.2 N hydrochloric acid for 3 min. The reactionmixture was then cooled to room temperature and extracted with ether (3X 25 ml.). After evaporation of the ether and residual colorless oil wasdissolved in 100 ml. benzene followed by addition of 2.04 g. (0.0138mol) of 2,3-bis-hydroxylamine-2,3-dimethylbutane. This mixture wasstirred for 45 min. Lead dioxide (20.0 g.) was then added with somecooling and the mixture was stirred for another 8 min. The deep redreaction mixture was filtered through Celite and evaporated to drynessin vacuo. The resulting red oil was chromatographed on silica withether. The red fraction was collected and evaporated in vacuo to yieldred crystals of the diester radical Vll. Recrystallization frompetroleum ether yielded 1.60 g. (33%) of VII, m.p. 7l.

Anal. Calculated for C H N O C; 55.9; H: 7.87; N: 8.16. Found: C: 55.84;H: 7.81; Ni 8.30.

Methyl- 1 1 ,3-dioxy-4,4,5 ,5-tetramethyl-4,5-dihydro-2-imidazomethyl)-malonic acid monoethyl ester VIII The diester radical VII(0.300 g.,.0.0087 mol) was stirred in 45 ml. 0.0215 N sodium hydroxidefor 18 hours at room temperature. The resulting solution wasconcentrated in vacuo to 5 ml. while maintaining the temperature below20. The remaining 5 ml. of solution was extracted with ether (3 X 15ml.) and the ether extracts washed with little water. The combined waterphases were passed through a column containing 5.0 g. of cation exchangeresin (Dowex 50W-X8). The column was eluted with ml. water, and theeluate was concentrated in vacuo as before. Two drops of 0.2 N HCl werethen added to the remaining water solution followed by contraction withchloroform (3 X 50 ml.).

The combined chloroform extracts were dried with magnesium sulfate,filtered and evaporated to dryness in vacuo. The residual red oil, ondrying overnight in vacuum over phosphorous pentoxide, yielded redcrystals of VIII 0.184 g. (67%), m.p. 60, (gas evolution at about 90).The crystals of VIII were very hygroscopic twice on silica with ether togive 0.425 g. (45%) of the but could be handled under dry nitrogen.Absorption radical XI as a red oil which did not crystallize. maximawere observed in the IR spectrum at KBR Anal. Calculated for C H N O C:57.6; H: 8.49; N: 1135, 1173, 1240, 1290 (max), 1370, 1445, 1600 10.33.Found: C: 57.44; H: 8.57; N: 10.27.

(broad), 1721, 2980 (M) and 3440 (M, broad). The 5 mass spectrum showedno molecular ion but an ion at -F -9 m/e 271 (M 44) was Observedlmldazole)-2-methylprop1on1c acid XII The next experimental workutilizes the reaction The radical XI (0.133 g., 0.000491 mol) wasstirred scheme as follows to make the product XII of this inin 43 ml.0.01 16 N sodium hydroxide for 5 hours, folvention. lowed by addition of0.3 ml. of 4.5 N sodium hydroxide SCHEME c 1) GH /H2O(C2I-I5O),CHCH2CHCOOC2I-I5 2 HEAT CH3 1x 1 NHOH o H@/H2O OHCCH2CHCOOC2H5I NHOH N CH3 /(JH2CHCOOC2H5 2 OXIDATION 1T -CH X 0. X1 9 GH /H O NCH2(|IHCOOH 1T1 CH: 0.

XII

Ethyl and stirring for another 20 min. The reaction mixture-3-(1,3-dioxy-4,4,5,5-tetramethyl-4,5-dihydro-2- a brought to P y adding1 N hydrochloric imidazole) 2 methy1propionate XI acid and extractedwith chloroform (4 X 100 ml.) The combined chloroform phase was driedwith magnesium The aldehyde ester X g-, 7 mol) was sulfate, filtered andevaporated to dryness in vacuo to stirred with2,3-bis-hydroxylamino-2,3-dimethylbutane give a red oil. This oil waschromatographed twice on (0.51 g., 0.00347 mol) in ml. benzene for min.at silica with a 3:2 mixture of methanol-ether. On recrysroomtemperature. The reaction mixture was then tallization fromether-petroleum ether the red crystalcooled at 10, lead dioxide (60 g.)was added and the line product melted at about d., (41 mg., 34%).

mixture stirred 10 min. The deep red reaction mixture 40 The followingexperimental work utilizes the reaction was filtered through Celite andevaporated to dryness scheme as follows to make the product XIV of thisin vacuo. The resulting red oil was chromatographed invention:

SCHEME D 1) PHENYLITHIUM 2) c1cH cH (oc n CH cn 2 3 HcH cHbc H 2 XIII 3)OXIDATION xIv 2-( 2-Pyridyl)-propionaldehyde diethylacetal XIII Asolution of 2-ethylpyridine (13.9 g., 0.13 mol) in 50 ml. of dry etherwas added dropwise during 15 min. to a solution of phenyllithium inbenzene (60 ml., 2 moles), with stirring under dry nitrogen. Thismixture was refluxed for 30 min. and chloroacetaldehyde diethylacetal(9.91 g., 0.0647 mol) was then added with continued heating for 5 hoursfollowed by stirring at room temperature for 12 hours. The brownreaction mixture was then poured over ice (50 g.), whereupon a brown oilseparated. The water phase was extracted with ether (3 X 100 ml.) andthe combined ether phases dried over potassium carbonate and evaporatedin vacuo. The residual brown oil gave on distillation the pyridylacetalXIII, b.p. 838510.05 mm. Hg, 7.0 g. (49%).

Anal. Calculated for C H NO C: 69.92; H: 9.48; N: 6.27. Found: C: 69.95;H: 9.28; N: 6.27.

-py yhp py l- 1 ,3-dioxy-4,4,5,5-tetramethyl-4,5-dihydroimidazole XIVThe acetal XIII (2.0 g., 0.00896 mol) was refluxed in 29 ml. of 0.031 Nhydrochloric acid. After 15 min. the solution became clear and was thencooled to room temperature and made alkaline with 1 N sodium hydroxide.An oil separated and the water phase was extracted with ether (3 X 50ml.). The combined ether extracts were dried with potassium carbonateand evaporated in vacuo.

To the resulting colorless oil was added a solution of2,3-bis-hydroxylamino-2,3-dimethylbutane (1.33 g.,

0.00896 mol) in 50 ml. ether. This mixture was stirred for 2'hours atroom temperature, and then evaporated to dryness in vacuo. The resultingoil was dissolved in 50 ml. of benzene and stirred with lead dioxide(13.3 g). for min. at room temperature. The mixture was then filteredover Celite, evaporated to dryness in vacuo and chromatographed oversilica with a 1:9:10 mixture of methanol-ethyl acetate-benzene. The deepred fraction containing the radical was evaporated to dryness, andrecrystallized from ether-petroleum-ether to give 0.97 g. (39%) of thepyridyl radical XIV, deep red crystals mp. 113-116.

Anal. Calculated for C H N O C: 65.2; H: 7.97; N: 15.21.

Found: C: 65.36; H: 7.71; N: 15.33.

The imino nitroxide may be prepared either directly from the1,3-dihydroxydiazole or indirectly from the nitronylnitroxide. Themethod of choice will depend upon the particular groups at the betacarbon atom or asymmetric center, The imino nitroxide may be preparedfrom the 1,3-dihydroxydiazole by the use of sodium nitrite, acid anddimethylformamide as solvent. Alternatively, the nitronylnitroxide maybe reduced by using a trialkylphosphite or other method disclosed in theaforementioned co-pending application.

If the spectrum of the unknown corresponds to the spectrum of a knownbuffer solution that is outside the i 1.0 pH range, only a crudeestimate of the pH is possible. If more precise results are required, adifferent indicator having a pK more closely corresponding to the pH ofthe unknown is then selected and the comparison with the known spectrarepeated. Several trials may be required to achieve optimum results.

Although the foregoing invention has been described in some detail byway of illustration for purposes of clarity-of understanding, it will beapparent to one skilled in this art that certain changes andmodifications may be practiced within the spirit of this invention aslimited only by the scope of the appended claims.

What is claimed is:

wherein R are the same of different and are lower alkyl of from 1 to 6carbon atoms; X is hydrogen or lower alkyl of from 1 to 6 carbon atoms;and Y is different from X and is lower alkyl, lower alkoxycarbonyl orcarboxylic acid. 2. An organic free radical in accordance with claim 1of the formula:

wherein X is hydrogen or lower alkyl of from 1 to 6 carbon atoms, Y isdifferent than X and is lower alkyl lower, alkoxycarbonyl or carboxylicacid.

2. An organic free radical in accordance with claim 1 of the formula: